Tay-Sachs or Sandhoff disease result from mutations that affect the Alpha- or Beta- polypeptide of the enzyme Beta-hexosaminidase, respectively. Both polypeptides are synthesized as precursors and undergo glycosylation, phosphorylation, association to limited proteolysis to give rise to the catalytically active enzyme that resides in lysosomes. Some of the mutations interfere with transport or catalytic activity of the respective polypeptide whereas others prevent synthesis. The aim of this proposal is to define these mutations precisely. Cloned and sequenced cDNAs encoding the two polypeptides will be used as guides to the amino acid sequence or as probes for messenger RNA. Structural changes that occur in the normal process of maturation and in the genetic diseases will be analyzed by radioactive microsequencing and peptide mapping, using polypeptides biosynthetically labeled in cultured fibroblasts or in cell-free translation. Emphasis will be on mutations that effect intracellular transport and processing; among them is a defect of the Alpha-polypeptide preventing its association with the Beta-, that appears to be common among the clinically milder Beta-hexosaminidase A deficiencies. For those mutations in which the polypeptide is not made at all (which include the well-known classic Tay-Sachs disease in patients of ashkenazi origin) structural and metabolic studies of messenger RNA will be pursued and related to the gene defect. The results should clarify the biochemical and clinical heterogeneity of the inherited Beta-hexosaminidase deficiency diseases, be useful for diagnostic and carrier tests in special instances, describe structure-function relationships of different domains of the polypeptides, and reveal functions that might otherwise remain hidden.